Submitted in complete fulfillment for the degree of Doctor of Philosophy (Food Science and Technology), Durban University of Technology, Durban, South Africa, 2016. / Bambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals. / D

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2018. / The aim of this study was to produce Bambara groundnut (BGN) starch-catechin complex using chemical initiators (ascorbic acid and hydrogen peroxide) and cyclodextrin (alpha and beta) with the view to obtain a functional ingredient for the food industry. BGN starch was successfully extracted from BGN flour through dry milling method, yielding 32% of BGN starch. Native BGN starch was chemically modified using ascorbic acid (1% w/w) and hydrogen peroxide (165% w/w) as redox, biocompatible initiator for grafting catechin to the BGN starch. In addition, cyclodextrin (alpha and beta) were also used as initiators for modifying BGN starch through complexation methods. Complexation methods used included the microwave, co-evaporation and kneading. The characterization of native and modified BGN starches was carried out by performing scanning electron microscopy, powder X-ray diffraction, Fourier Transform Infrared (FTIR) and fluorescence spectroscopy analysis. Functional, thermal and rheological properties of native and modified BGN starches were evaluated. The pasting properties of BGN starches were determined using the Rapid Visco Analyser (RVA). According to the SEM profile, native BGN starch had round, oval and elliptical shapes typical for legume starches. Native BGN starch displayed a typical type-C crystallinity which is common among legumes with strong peaks at 2θ of 15o, 17o and 23o. BGN starches modified through complexation methods had sharper peaks indicating an increase in starch crystallinity; however, following chemical modification there was loss in starch crystallinity which was evidenced by the amorphous region in the chemically modified BGN starches. Structure of native and modified BGN starches was confirmed by FTIR. The FTIR spectra of native BGN starch showed variable peaks at 3285.34 cm-1, 2931.69 cm-1, 1634.36 cm-1, 1336.77 cm-1 which are attributed to OH stretching, C-H stretching, water bending vibrations and C-O stretching, respectively. Furthermore, the FTIR results confirmed that native BGN starch is made up of glucose molecules just like all other starches. All modified BGN starches displayed a new absorption peak at 1020 cm-1 wavelength, thus indicating that starch modification was successful. On the other hand, all BGN starch-catechin complexes displayed a new absorption peak in the range of 1520 -1560 cm-1, attributed to the C-C stretching within the aromatic ring of the catechin. The successful grafting of catechin to BGN starch was also confirmed by the fluorescence spectroscopy results, where all the BGN starch-catechin complexes had an emission peak at 320 nm while native BGN starch had an emission peak at 270 nm. Antioxidant capacity of BGN starch was determined through DPPH and ORAC antioxidant assays. Within the DPPH assay, the antioxidant activity ranged from 2.26 to 38.31 μmol TE/g. The antioxidant activity of modified BGN starch-catechin complexes was significantly (p ≤ 0.05) higher than the ones modified without catechin. On the other hand, within the ORAC assay, the antioxidant activity ranged from 0.07 to 126.71 μmol TE/g. As opposed to the results obtained in DPPH assay, the antioxidant activity of chemically modified BGN starch-catechin complexes was significantly (p ≤ 0.05) higher than that of complexed BGN starch-catechin complexes. Chemical modification significantly increased the swelling capacity of native BGN starch while complexation methods significantly reduced it.